Linear growth deficiencies have multiple etiologies, ranging from injury and illness to genetic bone disease. Bone elongation disorders are particularly challenging to treat because of the relative inability to regulate molecular delivery o the growing skeleton. The primary obstacle to successful clinical intervention is lack of molecular delivery to the growing skeleton. The primary obstacle to successful clinical intervention is lack of methods for targeting therapeutics to growth plate cartilage, which does not have a penetrating blood supply. Existing procedures for limb lengthening involve invasive surgery or drug regimens, which are to date only partially effective. Data generated by the applicant show that localized heating increases molecular uptake in growth plate cartilage in vivo, suggesting that heat could be a noninvasive and inexpensive alternative for augmenting delivery of bone-lengthening drugs. The long-term goal is to identify physiological mechanisms underlying temperature-enhanced bone elongation in the growth plate. The overall objective of the problem-solving proposal is to determine whether heat augments the bone-lengthening effects of systemic growth regulators. Insulin-like growth factor (IGF)-I is a potent stimulator of linear growth and part of a drug regimen used in children. The central hypothesis, based on strong preliminary data and tested under two specific aims using dynamic in vivo multiphoton microscopy, is that heat localizes delivery of systemic IGF-I into growth plates to promote additive bone lengthening. Specific Aim 1 uses in vivo cartilage imaging to determine dose, timing, and temperatures that maximize IGF-I uptake in 5-week-old mouse tibial growth plates. Injections of fluorescently labeled IGF-I and size proxy tracers will be given to quantify molecular uptake and clearance in the growth plate. Real time imaging will be used to determine delivery rate and total volume of labeled molecules in the growth plate and surrounding vasculature after timed injections. Permeability of the vessels and matrix will be measured at different doses and temperatures. Specific Aim 2 uses a novel limb heating model to determine if heat-enhanced IGF-I uptake in growth plates causes unilateral limb lengthening, analyzed by quantifying growth rate and biomarkers of IGF-I activation in heat-treatable tibiae. Injections of IGF-I and its receptor antagonist will explicitly show if heat-enhanced uptake increases bone length. IGF-I deficient growth hormone receptor knockout mice will be used to test the drug-targeting efficacy of heat in a disease model. The rationale is to facilitate design of heat based drug-targeting approaches to enhance length at specific skeletal sites using noninvasive techniques. This project is innovative by using multiphoton imaging to assess growth plate physiology at the cellular level in vivo, in a dynamic way not possible with other methodologies. This contribution is significant because it can yield transformative findings that mechanistically link heat, bone lengthening, and vascular access to growth plates. Such results could fundamentally shift the approach that physicians take in treating a spectrum of growth plate disorders, leading to new therapies with better outcomes by reducing amount, toxicity and costs of high-dose systemic pharmaceuticals.